Large-scale bacterial expression and protein purification.
1. For expression of 6xHis-tagged proteins encoded on pET15b or pET29b(+) vectors in Lemo21(DE3) E. coli, either a single colony from an agar plate, 25.0 μL of overnight-grown culture, or 25.0 μL of a 20% glycerol culture stock is inoculated into 50.0 mL, 500 mL, or 1.00 L of lysogeny broth (LB) media or Terrific Broth II (TBII) media supplemented with 50.0 μg·mL-1 carbenicillin (pET15b) or 50.0-100.0 μg·mL-1 kanamycin (pET29b(+)) at final concentration.
2. To induce protein expression, cells are grown at 37°C shaking at 200-250 rotations per minute (rpm) overnight (16-22 hours) with 500 µM isopropyl β-D-thiogalactopyranoside (IPTG) final concentration added either prior to inoculation or 4 hours prior to harvesting cells.
3. Cells are harvested by centrifugation at 3,000-4,000xg for 10 minutes at 4°C.
4. Cell pellets are resuspended in 20.0-30.0 mL of either lysis buffer #1 supplemented with 1.00 mg·mL-1 phenylmethylsulfonyl fluoride (PMSF) and a small amount of deoxyribonuclease I (DNase I), lysis buffer #2, or lysis buffer #3.
5. Cells are lysed via either sonication at 40-70% amplitude, FastPrep-96 at 1,600 rpm with glass beads, or cell disruption using a microfluidizer at 18,000 psi.
6. Crude cell lysates are centrifuged at 24,000xg for 30-60 minutes at 4˚C.
7. To purify clarified cell lysates via gravity Ni-NTA immobilized metal affinity chromatography (IMAC), 2.00 mL of Ni-NTA agarose resin is pre-equilibrated with 10.0 mL of either lysis buffer #1, wash buffer #2, or wash buffer #3.
8. Clarified cell lysate is loaded onto the column and the flow-through is collected.
9. The column is washed 1-3 times with 20.0 mL of either wash buffer #1, wash buffer #2, or wash buffer #3.
10. The proteins are eluted from the column in 5.00-15.0 mL of either elution buffer #1, elution buffer #2, or elution buffer #3 and the eluates are collected.
11a. For keeping 6xHis tags intact, IMAC-purified proteins are buffer exchanged into high salt Tev cleavage buffer or low salt Tev cleavage buffer using Amicon Ultra-15 Centrifugal Filter Units. Proceed to step 14.
11b. For removing 6xHis tags, IMAC-purified proteins are concentrated to ~1.0 mL using Amicon Ultra-15 Centrifugal Filter Units, 1.0-5.0 mg·mL-1 6xHis-tagged Tobacco Etch Virus (TEV-6xHis) protease is added at a ~1:100 dilution, and the reaction is incubated at 25°C overnight. Proceed to step 12.
12. TEV-6xHis protease is removed via secondary Ni-NTA IMAC purification, eluting proteins in 5.00 mL of lysis buffer #1 or lysis buffer #2.
13. Cleaved protein is concentrated in Amicon Ultra-15 Centrifugal Filter Units.
14. Protein concentrations are measured using either a QuBit 2.0 and QuBit Protein Assay Kit or a NanoDrop 8000 with extinction coefficients predicted from amino acid sequence using the ProtParam tool2 or Biopython module3.
Size-exclusion chromatography (SEC).
1. IMAC-purified proteins are prepared at ≥1 mg·mL-1 and applied to a Superdex 75 Increase 10/300 GL column or Superdex 200 Increase 10/300 GL column on a LC 1200 Series high-performance liquid chromatography (HPLC) machine for separation of IMAC-purified proteins based on molecular size.
2. SEC-purified proteins are eluted in either high salt Tev cleavage buffer, Tris buffered saline (TBS), phosphate buffered saline (PBS), or Dulbecco's Phosphate-Buffered Saline without calcium or magnesium (DPBS) (Thermo Scientific).
3. Monomeric protein fractions are collected and concentrated using Amicon Ultra-15 Centrifugal Filter Units with 3 kDa cutoff.
4. Protein concentrations are measured as in step 14 of "Large-scale bacterial expression and protein purification."
Small-scale bacterial expression and protein purification.
1. 25.0 μL of overnight-grown cultures of Lemo21(DE3) E. coli are inoculated into each well of Nunc 2.0 mL DeepWell 96-well plates containing 1.00 mL of LB media supplemented with 50.0 μg·mL-1 carbenicillin (pET15b) final concentration, and plates are sealed with adhesive Breathe-easy film covers. Optionally, 2-8 replicate plates are prepared to optimize purified protein yields.
2. Cells are grown at 37°C and shaken at 1,200 rpm for 3-4 hours.
3. 500 µM IPTG final concentration is added to each well.
4. Protein expression is induced for 4 hours at 37°C shaken at 1,200 rpm.
5. Cells are pelleted at 2,272xg for 2-5 minutes in a plate centrifuge.
6. Pellets are resuspended in 50.0 μL of lysis buffer #1 supplemented with 1.00 mg·mL-1 PMSF and a small amount of DNase I, combining pellets of any replicate plates.
7. Cells are transferred to skirted 96-well polymerase chain reaction (PCR) plates and sealed with adhesive foil covers for cell lysis via a microplate sonicator at 40-60% amplitude for 2-4 minutes in an ice bath.
8. Crude cell lysates are centrifuged at 2,272xg for 5 minutes in a plate centrifuge.
9. Clarified cell lysates are applied to His MultiTrap HP 96-well plates pre-equilibrated in lysis buffer #1, and IMAC purification undergone following the manufacturer's protocol for purification except resin is washed three times in wash buffer #1 and eluted three times, each time using 50.0 μL of elution buffer #1.
10. Eluates are consolidated into a single 96-well PCR plate.